Inflammation is a major component in the pathogenesis of brain injury during stroke. Cell signaling pathways prominently involved in the inflammatory cascade are initiated through Toll-like receptors (TLRs). Thus TLRs may be novel targets for stroke therapeutics. TLR4 is responsible for ischemic tolerance induced by systemic administration of lipopolysaccharide (LPS). Potential deleterious side effects preclude translation. We have found that additional TLRs (TLR7 & TLR9) are also potent targets to induce preconditioning against stroke. Pretreatment with the TLR9 agonist, CpG ODNs, reprograms cell signaling during subsequent stroke and the resultant inflammatory cell signaling is changed to potent neuroprotection. CpG ODNs are well tolerated in humans offering rapid translation as pre-stroke treatment for patients at high risk (e.g. new TIA, pending CABG surgery). Here we propose to characterize this novel prophylactic stroke therapy and demonstrate the efficacy and immune cell activation in the setting of stroke. We will address the potential mechanisms that underlie neuroprotection and whether these mechanisms act systemically and/or are located in the CNS as human treatments may optimally be directed systemically or centrally. Aim 1. Characterization of neuroprotection induced by the TLR9 agonist, CpG ODNs. Aim 3. Determine whether the primary inducers and effectors of LPS preconditioning are shared by imiquimod and CpG preconditioning pathways. Aim 2. Determine the relative contribution of CNS resident cells and systemic hematopoietic cells to TLR9 induced neuroprotection. Aim 3. Determine whether TNFa and IFNb are critical effectors of ischemic tolerance elicited by TLR9 (CpG) preconditioning. Aim 4. Determine whether CpG preconditioning reprograms the response to stroke through modulation of TLR signaling pathways.